The perilipins are a family of proteins, first identified in this laboratory, expressed only in adipocytes and in steroidogenic cells, where they coat the surfaces of intracellular neutral lipid droplets. Our evidence suggests a role for these proteins in both lipid hydrolysis and packaging. Lipid hydrolysis is initiated by protein kinase A-mediated phosphorylation hormone-sensitive lipase (HSL) in adipocytes and the closely related cholesteryl esterase in steroidogenic cells. Lipolytic stimulation leads to translocation of HSL from the cytosol to the lipid droplet surface and simultaneous dispersal of perilipin from the droplet surface. From such data, we speculate that perilipins may provide a barrier to prevent inappropriate hydrolysis by HSL in unstimulated adipocytes. Evidence for a role for perilipins in lipid packaging includes the following: 1) The smallest detectable adipocyte lipid droplets are bounded by perilipin; 2) Expression of ectopic perilipin in non-adipocyte cells results in the appearance of small lipid droplets that are coated with perilipin, indicating that perilipin can drive lipid droplet formation; and 3) Ablation of perilipins in differentiating 3T3-L1 adipoblasts with a retroviral antisense construct prevents lipid droplet formation, but not expression of other adipocyte-specific markers. Further experiments have revealed the presence of other proteins that are intrinsic to lipid droplets in adipocytes and steroidogenic cells. One example, ADRP (adipocyte differentiation- related protein), shares a short sequence of significant similarity with perilipin and is found only on the smallest detectable lipid droplets in adipocytes, suggesting a role in nascent droplet formation. Further, steroidogenic cells, which contain extremely small lipid droplets compared to adipocytes, express higher levels of ADRP than perilipin. The finding that two separate gene products, perilipins and ADRP, are located exclusively on lipid droplet surfaces provides compelling evidence that these droplets are unique structures composed of a specific complement of proteins that dictate their formation and metabolism. Sequence analysis of additional proteins we have isolated should result in a clear definition of the components of the lipid droplets, and the yeast two-hybrid system with an adipocyte fusion library is being used to uncover interactions among proteins involved in droplet construction and lipid metabolism.